David D. Weis

Extraction of average masses of deuterated peptides from LC-MS data in hydrogen exchange-mass spectrometry (HX-MS) is a challenging task that is not well-suited to traditional LC-MS feature extraction algorithms. While commercial software is available, no feature extraction algorithms specific to HX-MS data analysis have been fully described in the literature. The present work describes such an algorithm. hxsparse uses an exact model that describes the deuteration of each peptide in terms of linear combinations of discrete deuteration states. Sparsity is introduced into the model using least absolute shrinkage and selection operator (LASSO) regularization. We demonstrate that automated feature extraction with hxsparse outperforms commercial software and nearly matches the results obtained from expert curation of HX-MS data.

Ubiquitin-like modifier activating enzyme 5 (UBA5), an E1 enzyme in the UFMylation pathway, is of interest as a therapeutic target in diseases such as cancer due to the critical role that UFMylation plays in cellular function. Strategic structure–activity relationship optimization of high-throughput screen hit 6, a Bruton’s tyrosine kinase covalent inhibitor, was conducted by using both conventional and microscale library synthesis. This approach identified 49 and 50 as potent and selective noncovalent UBA5 inhibitors with significantly improved surface plasmon resonance and nanodifferential scanning fluorimetry biophysical profiles. Cellular target engagement for 49 and 50 was confirmed by a cellular thermal shift assay and translated to inhibition of UFMylation of UBA5 and UFC1 in retinal pigment epithelial-1 cells as well as reduction of UFMylation of the downstream protein UFMylation substrate RPL26. Furthermore, 49 and 50 demonstrated specificity for UBA5 among the other E1–E2 transesterification pathways. In this communication, we report the discovery and synthesis of potent and selective UBA5 inhibitors, providing valuable tool compounds for studying the UFMylation pathway.

An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.

Hydrogen exchange-mass spectrometry (HX-MS) is a valuable analytical technique that can provide insight into protein interactions and structure. The deuterium labeling necessary to gain this insight is affected by many physical and chemical factors, making it challenging to achieve high reproducibility. Poor precision during dispensing, transfer, and mixing of solutions during the experiment contributes substantially to the overall variability. While the use of a robotic liquid handler can potentially improve precision, its operation must be optimized. We observed poor precision in data collected using a robotic liquid handler to perform HX-MS. In this work, we describe how we were able to improve that system's precision considerably based on tracking performance using caffeine, caffeine-d(3), and caffeine-d(9) as tracers for the sample, label, and quench to report on each operation of the liquid handling workflow. The insights gained about liquid handler performance and the three-tracer approach can aid in optimizing HX-MS workflow operations, whether performed manually or when using a liquid handling system. Additionally, these tracers can be incorporated as internal tracers during an experiment to report on the labeling and quench operations of each sample throughout the run and, if desired, be used to implement an uptake correction described previously.

319‐44 is a human monoclonal antibody capable of passively protecting mice against tick‐mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319‐44 has complement‐dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å‐resolution crystal structure of 319‐44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first‐generation Lyme disease vaccine approved in the United States. The 319‐44 epitope is focused on OspA β‐strands 19, 20, and 21, and the loops between β‐strands 16‐17, 18‐19, and 20‐21. Contact with loop 20‐21 explains competition with LA‐2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first‐generation Lyme disease vaccine clinical trials. A high‐resolution B‐cell epitope map of OspA will accelerate structure‐based design of second generation OspA‐based vaccines.

In this paper, we introduce a screening protocol for epitope mapping by hydrogen exchange mass spectrometry (HX-MS) that has higher throughput than a traditional HX-MS epitope mapping. In the screening protocol, three HX labeling times (20, 1000, and 86400 s) are each measured without replicates. The experimental protocol is anchored on a single epitope mapping experiment conducted using the traditional complete protocol (five HX times measured in triplicate) that is used to define HX times and define significance limits. Previously, we reported traditional epitope mapping results on the Borrelia burgdorferi outer surface protein A (OspA) antigen that are in excellent agreement with the X-ray crystallography results. Here, we show that the screening protocol and complete HX-MS identify identical epitopes of OspA but that the screening protocol has a 5-fold higher throughput.

The Lyme disease (LD) vaccine formerly approved for use in the United States consisted of recombinant outer surface protein A (OspA) from Borrelia burgdorferi sensu stricto (ss), the bacterial genospecies responsible for the vast majority of LD in North America. OspA is an similar to 30 kDa lipoprotein made up of 21 antiparallel fl-strands and a C-terminal alpha-helix. In clinical trials, protection against LD following vaccination correlated with serum antibody titers against a single epitope near the C-terminus of OspA, as defined by the mouse monoclonal antibody (MAb), LA-2. However, the breadth of the human antibody response to OspA following vaccination remains undefined even as next-generation multivalent OspA-based vaccines are under development. In this report, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes recognized by a unique panel of OspA human MAbs, including four shown to passively protect mice against experimental B. burgdorferi infection and one isolated from a patient with antibiotic refractory Lyme arthritis. The epitopes grouped into three spatially distinct bins that, together, encompass more than half the surface-exposed area of OspA. The bins corresponded to OspA fl-strands 8-10 (bin 1), 11-13 (bin 2), and 16-20 plus the C-terminal alpha-helix (bin 3). Bin 3 was further divided into sub -bins relative to LA-2's epitope. MAbs with complement-dependent borreliacidal activity, as well as B. burgdorferi transmission-blocking activity in the mouse model were found within each bin. Therefore, the resulting B cell epitope map encompasses functionally important targets on OspA that likely contribute to immunity to B. burgdorferi.